The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the relatively small vascular effects of NO in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. We also find that NO-bioactivity destruction appears to occur in tissues to a much greater extent than in the vascular bed. We have also been fortunate in being able to use the Critical Care Medicine Department canine facility and test inhaled nitrite and nitrate ions in hypoxic dogs to see if administering NO bioactivity in this way would be therapeutically possible. We tested nitrate as there is evidence that pulmonary tissue has some levels of enzymes, such as xanthine oxido-reductase that can reduce nitrate ions to nitrite and NO. We have found strong effects of nitrite inhalation on both pulmonary and systemic parameters, as has been reported for other animal models, but the effects of nitrate are only seen intermittently, suggesting as it is known that nitrate in the dog salivary fluid is reduced to nitrite and then systemic and pulmonary exposure give NO effects due to subsequent, expected nitrite reduction processes. These results have just been published. We have also continued our studies on dietary variations in nitrate and nitrite in rodent diet ingestion. These dietary and genetic manipulations of rodents has shown that NOS 1 (nNOS) and myoglobin knockouts have markedly reduced levels of skeletal muscle nitrate suggesting that, as we predicted, both are involved in the high levels in muscle. However, we also find that dietary limitations of nitrate and nitrite lower these levels greatly (more than in blood or liver and more than many genetic manipulations) and that return of these ions to the diet results in rapid accumulation and, indeed, in some cases an overshoot of the levels. Detailed metabolic studies of nitrate-nitrite-NO metabolism in animals and people require tracer studies which may be approached using non-toxic heavy isotopes. Our collaborators in Newcastle are developing protocols to do this in animals and now in human subjects. They have sent us samples from various animal ingestion studies and we are using our highly sensitive and accurate chemi-luminescence methods to quantity nitrate and nitrite levels in these blood samples. (One of their staff visited our laboratory for several weeks to learn how to do these assays.) Early human studies have examined the effects of ascorbic acid on dietary nitrate and nitrite absorption. Our collaborators in Sao Paulo, Brazil have much experience in the pharmacology of NO and we are working with them on various animal models of hypertension with the hope of eventual clinical studies. Our collaborators in Bangkok have administered low levels of nitrite to thalassemia patients and we have worked with them to show that VASP phosphorylation is affected, as a parameter for measuring platelet inhibition (as described in DK 025093). Most encouraging, we have established a robust collaboration with a muscle physiology group in Exeter, UK and we have been measuring the levels of nitrate at rest and with exercise in the human tissues and have confirmed that our animal results obtain for human subjects, as other groups have now reported. We believe that muscle levels of nitrate must be considered in dietary interventions, for pharmacological effects or for affecting performance. These results have also been recently published. Two new projects have very recently been initiated. First, we are studying the mechanism of nitroglycerine reduction as our recent work has suggested the possibility of new pathways, including reduction by salivary bacteria, that might enhance the clinical utility of this classic agent. Second, we have been measuring nitrate and nitrite in whole rodent eyes as a beginning of measuring these levels in various compartments of the eye in larger animal models and perhaps in human samples. Early results suggest that the nitrate-nitrite-NO reduction pathway is also very important in eye NO physiology and perhaps pathophysiology.